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Review of Cleanroom Microflora Introduction Cleanrooms and environmental monitoring Contamination sources Contamination control Tim Sandle The human microbiome and the microbial ecology of www.pharmamicro.com people Case study: Microorganisms are found in cleanrooms Why cleanroom microflora matters http://www.pharmamicro.com http://www.pharmamicro.com Cleanrooms Cleanrooms: highly controlled environments with defined air quality Required for surgical units, hospital pharmacies and Cleanrooms, cleanroom design, contamination sources, aseptic drug manufacture. environmental control and environmental monitoring Airborne particulate control Air movement and air direction, HEPA filters, Temperature and humidity control, Cleaning and disinfection, Staff behaviours and gowning. Certified to meet ISO 14644. Operated to meet EU GMP. http://www.pharmamicro.com http://www.pharmamicro.com Contamination sources Contamination sources People The body harbours over thirty million bacteria The average person sheds 1,000,000,000 skin cells per day 10% have micro-organisms on them Particles are released from people via the mouth and nose (minute liquid droplets) ~ extremely contaminated with microorganisms. http://www.pharmamicro.com http://www.pharmamicro.com Contamination sources Contamination control #1 People Control: The rate of particle deposition varies according to Personnel wear suitable speed and activity. cleanroom clothing. When stationary, people generate approximately Good aseptic practices: 100,000 particles of 0.3 µm or greater Slow, deliberate walking Aseptic technique When walking slowly (2 miles per hour), people shed around 5 million particles When walking briskly (5 miles per hour), people shed around 7.5 million particles. http://www.pharmamicro.com http://www.pharmamicro.com Contamination control #2 Environmental Monitoring Air filtration Environmental monitoring is different to UDAF devices environmental control . Barrier technology: Cleanrooms and clean areas are assessed through an Isolators and RABS environmental monitoring programme. Water of low bioburden Monitoring: Keeping areas clean and To assess cleanliness. dry Physical assessment of airborne particles. Cleaning and disinfection Checks on temperature, humidity, pressure etc. Sterilisation Collection of data relating to the numbers and types of Single-use disposable microorganisms present on surfaces, in the air and products from people. http://www.pharmamicro.com http://www.pharmamicro.com Microbiological environmental monitoring The classic techniques: Active air-sampling: volumetric air- sampler Passive air-sampling: settle plates Surface samples: contact (RODAC) plates and swabs Personnel samples: Finger plates and gown plates Methods highly variable. New generation of ‘real time’ viable air samplers. http://www.pharmamicro.com http://www.pharmamicro.com Microorganisms in cleanrooms Microorganisms recovered Microbiologists count the number of Variables affect the types of microorganisms ‘colony forming units’ (cfu) and recovered: identify the contaminants. Monitoring methods, To assess: Location of the monitoring sample, Cleanroom operation issues Types of agars, Product risk investigations Disinfectant neutralisers, Cleaning and disinfection issues. Incubation temperature, Aseptic practices. Incubation time, Identification method. http://www.pharmamicro.com http://www.pharmamicro.com Human Microbiome Project The Human Microbiome Project (2008) Dedicated to the types and variations of microorganisms in relation to people (human microbiome). Microbiome: “the totality of microbes, their genetic elements (genomes), and environmental interactions in a particular environment ”. The human body contains over 10 times more microbial cells than human cells: 10 12 bacteria on the skin, 10 10 in the mouth, 10 14 in the gastrointestinal tract Weighing 200g http://www.pharmamicro.com http://www.pharmamicro.com Microbial ecology of people Microbial ecology of people Many of the species of microorganisms that form the microbiota are unknown. Advanced through genotypic microbial identification techniques (16s rRNA genes in lysed microbial DNA). Now understood that: Considerable diversity of species Variation between different locations on the body Variation between individuals Our microbiome changes over time. http://www.pharmamicro.com http://www.pharmamicro.com Microbial ecology of people Microbial ecology of people Different sites on the body have different microbial communities Based on nutritional and physicochemical requirements Liebig’s law of the minimum Microbial numbers are determined by the nutrients present Shelford’s law of tolerance Non-nutritional factors governing growth, such as pH and temperature Also antimicrobial factors and mechanical removal factors, like skin shedding http://www.pharmamicro.com http://www.pharmamicro.com Microbial ecology of people Microbial ecology of people Human gastrointestinal tract Age ~500 to 1000 species of bacteria live in the human gut Host genotype Eyes Gender Lower variety compared with other parts of the human body. Males have a high density of bacteria on their skin than females. The lachrymal glands continuously secrete bactericides such as lysozyme. Oral A diverse, abundant and complex microbial community. Bacteria accumulate on both the hard and soft oral tissues in biofilms. http://www.pharmamicro.com http://www.pharmamicro.com Microbial ecology of people Microbial ecology of people Skin #1 Skin #2 2 Average adult has 1.8m of skin Cleanroom risk Regional variation : With the skin epithelial cells are continually being shed Temperature range 25-37 oC so many microbial communities on the external surface Warmest area under the arms (36.6 oC) and the coolest area the fingers (29.5 oC). are unstable. pH of the skin varies between acidic and neutral, Forehead: pH 4.8 and under the arm: pH 6.9. Moisture High moisture: axilla, groin, and areas between the toes = high population density Other areas are relatively dry http://www.pharmamicro.com http://www.pharmamicro.com Microbial ecology of people Microbial ecology of people Skin #3 Skin #4 Most relate to 4 phyla: Actinobacteria: Gram- Why so many Gram-positive bacteria? positive bacteria e.g. Micrococcus, Corynebacteria Dry environment with high osmotic pressure. and Propionibacteria Gram-positive bacteria better adapted (resistant to Firmicutes : Gram-positive bacteria e.g. Clostridia and desiccation). Bacillus However, certain occluded regions (such as toe webs), Proteobacteria: Gram- negative bacteria e.g. have more variation, including Gram-negative bacteria. Escherichia, Salmonella, Vibrio, Helicobacter Bacteroidetes: Other Gram- negative bacteria http://www.pharmamicro.com http://www.pharmamicro.com Microbial ecology of people Microbial ecology of people Skin #5 Skin #6 So, different parts of the body lead to variation Variation between people: Region Environment Species Forehead & scalp Sebaceous glands Dominated by propionibacteria Ecologically, sebaceous areas had greater species and some corynebacteria richness than moist and dry one. Toe webs Occluded: increased moisture High microbial density: and temperature corynebacteria, brevibacteria, The areas with least similarity between people: fungi and Acinetobacter spp. spaces between fingers, the spaces between toes, axillae, and Arms and legs Dry Low microbial density: umbilical cord stump. staphylococci and micrococci. The areas with most similarity between people: Sole of foot High moisture due to shoes Many staphylococci and socks. beside the nostril, nares (inside the nostril), and on the back. Main body Varied Corynebacteria, fungi and Acinetobacter spp. http://www.pharmamicro.com http://www.pharmamicro.com Microbial ecology of people Skin #7 The most frequent species are: Bacteria: Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Corynebacterium spp ., and Acinetobacter johnsonii . Some anaerobic bacteria, like Propionibacterium , in hair follicles. Fungi: Yeasts Candida albicans, Rhodotorula rubra, Torulopsis , Microsporum gypseum , Trichophyton and fungi Rhizopus Trichosporon, Fusarium, Aspergillus and Penicillium species http://www.pharmamicro.com http://www.pharmamicro.com Why do microorganisms survive in cleanrooms? Varies depending upon the species and different factors: Temperature Available water A case study Concentration of organic compounds Concentration of hydrogen ions Concentration of inorganic compounds Concentration of particles in the air Redox potential Pressure Light intensity Geographical location and habitat http://www.pharmamicro.com http://www.pharmamicro.com What is found in cleanrooms? What is found in cleanrooms? Description of the human microbiota does not Variations also relate to: necessarily predict the types of microorganisms found Microbial identification method in cleanrooms. Phenotypic or genotypic Databases Variations relating to: Size, orientation (clinical or industrial) Cleanroom types and uses Periodic reclassification of microorganisms Grade, temperature and humidity Limitations of EM methods Geographical location EM agar and incubation regime Gowning requirements Culturability Certain locations of the body are more likely to release Some research suggest that less than 10% of bacteria found in organisms than others. cleanrooms are culturable Seasonality and time of sampling http://www.pharmamicro.com http://www.pharmamicro.com
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